Previously we have demonstrated that met-enkephalin-arg-phe (YGGFMRF) is metabolized by dipeptidyl carboxypeptidase and aminopeptidase. With the cooperation of Dr. S. Blumberg from the Weizmann Institute of Science, Rehovot, Israel, we have cointinued to search for effective enzyme inhibitors to block the YGGFMRF inactivation. Three mercaptoacylated dipeptides, HS-CH2-CO-Leu-Phe, HS-CH2-CO-Phe-Leu and HS-CH2-CO-Leu-D-Phe were provided by Dr. Blumberg as potential enzyme inhibitors. HS-CH2-CO-Leu-Phe effectively inhibited the inactivation of YGGFMRF by dipeptidyl carboxypeptidase in vitro. To estimate the in vivo effect of this inhibitor, the recovery of YGGFMRF released by substance P into spinal cord subarachnoidal space was measured. HS-CH2-CO-Leu-Phe, when added into the perfusion medium, was found to increase the release of YGGFMRF. Interestingly, the recovery of YGGFMRF released into spinal cord was increased by subcutaneous injection of CH3-CO-S-CH2-CO-Leu-Phe-OCH3. Whether this inhibitor is capable of reaching CNS after subcutaneous injection still remains to be further studied.